1. Technical Field
The field of this invention is proteomic assays.
2. Background
As more is learned about cellular pathways, the interactions of microorganisms with mammals, the growth and development of organisms, both prokaryotic and eukaryotic, there is an increasing need for improved methods of identifying protein members involved with these various processes. Also, as proteins and their function are identified, there is an increasing interest in being able to modulate the activity of proteins associated with diseased states. By screening for active compounds that modulate the activity, one is interested, not only in identifying the effect of the compound on the target protein, but also any effects the compound may have on other proteins present in the cell or medium.
There are numerous ways to separate components of a complex mixture based on differences in the components of mass, mass-to-charge, conformation, etc. Gel electrophoresis has been a major methodology for analyzing mixtures of proteins. Native proteins and denatured proteins are readily separated by their migration rate, which for the most part can be related to their molecular weight. Numerous techniques have been developed for identifying the separated protein bands. The Western blot was developed to identify proteins. As part of the Western blot, the proteins are separated by electrophoresis in a gel, followed by transfer of the protein bands to a membrane. The bands are then developed by various techniques, such as labeled antibodies or a non-specific protein dye. Other methods of separation include chromatography, e.g. HPLC, FPLC, capillary electrophoresis, etc.
The Western blot methodology has many desirable properties for identifying proteins in a mixture. However, even with 2D protocols, where one is dealing with a lysate or comparable mixture, there can be protein overlap, difficulties in detecting low amounts of a component, inadequate separations, inability to detect modifications of the protein and the like.
Today, one is interested in determining many aspects of the life of a protein in a cell, particularly in response to a change in the environment, e.g. a candidate compound as a drug. Proteins can undergo numerous modifications, such as partial or total degradation, glycosylation, ubiquitination, phosphorylation or dephosphorylation, acetylation, acylation, prenylation, etc. Being able to discern the existence of different aspects of the same protein is important in understanding pathways, such as identifying the major and minor status of the parent protein and the modified protein, where the effect of a candidate compound can be analyzed. Also, by being able to observe patterns of variations in the protein analogs and partial degradation products, one can identify cellular pathways and their interaction with other proteins.
There is, therefore, substantial interest in developing techniques that will allow the efficient investigation of cellular proteins, the effect of changes in the cellular environment on the protein, and being able to detect low levels of a protein of interest in the presence of large amounts of other proteins that may have analogous migration aptitudes.
Relevant Literature
U.S. Pat. Nos. 6,680,208 and 6,677,128 and WO94/29725 describe various Western blotting techniques. PCT/US02/27497 describes the use of fusion proteins employing enzyme fragmentation complexes for determining the status of the fusion protein in a cell.